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目的在大肠杆菌中表达耐草甘膦基因G6,并对其进行免疫反应性分析。方法将已构建好的携带G6基因的重组质粒pET28a-G6转化到大肠杆菌BL21中诱导表达,分别从诱导温度、异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度及诱导时间进行了表达条件的探索。获得的重组蛋白在非变性条件下镍亲和层析纯化后,经质谱鉴定,对其免疫反应活性进行测定,分析该原核表达蛋白与转G6基因水稻中蛋白的等同性。结果利用原核表达系统成功实现了G6重组蛋白的高效表达,其最适条件为:0.5 mmol/L IPTG、30℃和160 r/min摇床转速诱导7 h。质谱鉴定为5-烯醇式丙酮酸-莽草酸-3-磷酸合成酶(EPSPS)。Western blot鉴定后,表达的重组蛋白与转G6基因水稻中的G6蛋白有相同的免疫反应性。结论利用原核表达系统表达的G6重组蛋白可以替代植物外源蛋白进行转G6基因产品的食用安全性评价,也可用于转G6基因产品ELISA检测的抗体制备。
Objective To express glyphosate resistant gene G6 in Escherichia coli and analyze its immunoreactivity. Methods The constructed recombinant plasmid pET28a-G6 carrying G6 gene was transformed into E. coli BL21 for expression induction. The induction temperature, the concentration of IPTG and the induction of IPTG Time to explore the conditions of expression. The obtained recombinant protein was purified by nickel affinity chromatography under non-denaturing conditions and identified by mass spectrometry. The immunoreactivity of the recombinant protein was determined, and the identity between the prokaryotic protein and the G6 transgenic rice protein was analyzed. Results The efficient expression of recombinant G6 protein was achieved by using prokaryotic expression system. The optimum conditions were as follows: 0.5 mmol / L IPTG, 7 h at 30 ℃ and 160 r / min. Mass spectrometry was identified as 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS). After identification by Western blot, the expressed recombinant protein has the same immunoreactivity as the G6 protein in transgenic G6 rice. Conclusion The recombinant G6 protein expressed in prokaryotic expression system can replace the foreign protein of plant to evaluate the food safety of G6 gene product, and can also be used for antibody preparation of G6 gene product by ELISA.